Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins

Abstract Background The global demand for functional proteins is extensive, diverse, and constantly increasing. Medicine, agriculture, and industrial manufacturing all rely on high-quality proteins as major active components or process additives. Historically, these demands have been met by microbia...

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Main Authors: Jennifer A. Schmidt, Lubna V. Richter, Lisa A. Condoluci, Beth A. Ahner
Format: Article
Language:English
Published: BMC 2021-02-01
Series:Biotechnology for Biofuels
Subjects:
Online Access:https://doi.org/10.1186/s13068-021-01893-2
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spelling doaj-5b38b236796049bf8e368e80782315102021-02-14T12:48:11ZengBMCBiotechnology for Biofuels1754-68342021-02-0114111410.1186/s13068-021-01893-2Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteinsJennifer A. Schmidt0Lubna V. Richter1Lisa A. Condoluci2Beth A. Ahner3Department of Biological and Environmental Engineering, Cornell UniversityDepartment of Biological and Environmental Engineering, Cornell UniversityDepartment of Biological and Environmental Engineering, Cornell UniversityDepartment of Biological and Environmental Engineering, Cornell UniversityAbstract Background The global demand for functional proteins is extensive, diverse, and constantly increasing. Medicine, agriculture, and industrial manufacturing all rely on high-quality proteins as major active components or process additives. Historically, these demands have been met by microbial bioreactors that are expensive to operate and maintain, prone to contamination, and relatively inflexible to changing market demands. Well-established crop cultivation techniques coupled with new advancements in genetic engineering may offer a cheaper and more versatile protein production platform. Chloroplast-engineered plants, like tobacco, have the potential to produce large quantities of high-value proteins, but often result in engineered plants with mutant phenotypes. This technology needs to be fine-tuned for commercial applications to maximize target protein yield while maintaining robust plant growth. Results Here, we show that a previously developed Nicotiana tabacum line, TetC-cel6A, can produce an industrial cellulase at levels of up to 28% of total soluble protein (TSP) with a slight dwarf phenotype but no loss in biomass. In seedlings, the dwarf phenotype is recovered by exogenous application of gibberellic acid. We also demonstrate that accumulating foreign protein represents an added burden to the plants’ metabolism that can make them more sensitive to limiting growth conditions such as low nitrogen. The biomass of nitrogen-limited TetC-cel6A plants was found to be as much as 40% lower than wildtype (WT) tobacco, although heterologous cellulase production was not greatly reduced compared to well-fertilized TetC-cel6A plants. Furthermore, cultivation at elevated carbon dioxide (1600 ppm CO2) restored biomass accumulation in TetC-cel6A plants to that of WT, while also increasing total heterologous protein yield (mg Cel6A plant−1) by 50–70%. Conclusions The work reported here demonstrates that well-fertilized tobacco plants have a substantial degree of flexibility in protein metabolism and can accommodate considerable levels of some recombinant proteins without exhibiting deleterious mutant phenotypes. Furthermore, we show that the alterations to protein expression triggered by growth at elevated CO2 can help rebalance endogenous protein expression and/or increase foreign protein production in chloroplast-engineered tobacco.https://doi.org/10.1186/s13068-021-01893-2Recombinant proteinCellulaseTobaccoChloroplast-engineeringGibberellic acidRubisco
collection DOAJ
language English
format Article
sources DOAJ
author Jennifer A. Schmidt
Lubna V. Richter
Lisa A. Condoluci
Beth A. Ahner
spellingShingle Jennifer A. Schmidt
Lubna V. Richter
Lisa A. Condoluci
Beth A. Ahner
Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins
Biotechnology for Biofuels
Recombinant protein
Cellulase
Tobacco
Chloroplast-engineering
Gibberellic acid
Rubisco
author_facet Jennifer A. Schmidt
Lubna V. Richter
Lisa A. Condoluci
Beth A. Ahner
author_sort Jennifer A. Schmidt
title Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins
title_short Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins
title_full Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins
title_fullStr Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins
title_full_unstemmed Mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins
title_sort mitigation of deleterious phenotypes in chloroplast-engineered plants accumulating high levels of foreign proteins
publisher BMC
series Biotechnology for Biofuels
issn 1754-6834
publishDate 2021-02-01
description Abstract Background The global demand for functional proteins is extensive, diverse, and constantly increasing. Medicine, agriculture, and industrial manufacturing all rely on high-quality proteins as major active components or process additives. Historically, these demands have been met by microbial bioreactors that are expensive to operate and maintain, prone to contamination, and relatively inflexible to changing market demands. Well-established crop cultivation techniques coupled with new advancements in genetic engineering may offer a cheaper and more versatile protein production platform. Chloroplast-engineered plants, like tobacco, have the potential to produce large quantities of high-value proteins, but often result in engineered plants with mutant phenotypes. This technology needs to be fine-tuned for commercial applications to maximize target protein yield while maintaining robust plant growth. Results Here, we show that a previously developed Nicotiana tabacum line, TetC-cel6A, can produce an industrial cellulase at levels of up to 28% of total soluble protein (TSP) with a slight dwarf phenotype but no loss in biomass. In seedlings, the dwarf phenotype is recovered by exogenous application of gibberellic acid. We also demonstrate that accumulating foreign protein represents an added burden to the plants’ metabolism that can make them more sensitive to limiting growth conditions such as low nitrogen. The biomass of nitrogen-limited TetC-cel6A plants was found to be as much as 40% lower than wildtype (WT) tobacco, although heterologous cellulase production was not greatly reduced compared to well-fertilized TetC-cel6A plants. Furthermore, cultivation at elevated carbon dioxide (1600 ppm CO2) restored biomass accumulation in TetC-cel6A plants to that of WT, while also increasing total heterologous protein yield (mg Cel6A plant−1) by 50–70%. Conclusions The work reported here demonstrates that well-fertilized tobacco plants have a substantial degree of flexibility in protein metabolism and can accommodate considerable levels of some recombinant proteins without exhibiting deleterious mutant phenotypes. Furthermore, we show that the alterations to protein expression triggered by growth at elevated CO2 can help rebalance endogenous protein expression and/or increase foreign protein production in chloroplast-engineered tobacco.
topic Recombinant protein
Cellulase
Tobacco
Chloroplast-engineering
Gibberellic acid
Rubisco
url https://doi.org/10.1186/s13068-021-01893-2
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